Dendrimers are globular macromolecules of monodisperse distribution and size, in which all links emerge radially from a focal point or nucleus with a regular branching pattern and with repetitive units that contribute to one point of the branch. Each layer between the branching points is called generation and they are listed from the center out towards the periphery of the dendrimer.
It is important to first establish the nucleus, the number of generations, and the repetitive units used in each generation, to determine the size, the form, and the functional groups present.
In practice, Dendrimers are formed by repetitive units of the ABx type, where A and B are different functional groups and x is the amount of the B groups, a feature called multiplicity, which results in a uniform structure between the generations thereof, and have the sole purpose of increasing the amount of terminal functional groups in a regular manner and of geometric growth in amount and dependent upon their multiplicity as show in “Dendritic molecules: concepts, synthesis, perspectives.” of G. R. Newkome, C. N. Moorefield, F Vögtle; Weinheim, N.Y., VCH, 1996.
Dendrimers are generally obtained via synthesis with iterative stages using divergent synthesis, in which growth of the dendrimer begins from the nucleus out to the periphery (Tomalia et al., U.S. Pat. Nos. 4,435,548; 4,507,466; 4,558,120; 4,568,737; 5,338,532), or the strategy of the convergent growth, in which the synthesis of the final structure begins in the periphery via the construction of the different branches of the dendrimer, called dendrons, and as the last step the addition of these dendrons to the nucleus (Hawker et al., U.S. Pat. No. 5,041,515).
The increase in the number of terminal groups in a dendrimer is consistent with the equationZ=NCNbG Where:Z represents the number of terminal functional groups of the dendrimer.Nc represents the multiplicity of the nucleus (For example, Nc=3 for the ammonia and Nc=4 for Ethylene Diamine (EDA).Nb represents the multiplicity of branching.G represents the number of the generation of the dendrimer.For different multiplicities of the branching element, the total number of terminal functional groups is given by the equation:
  Z  =            N      c        ⁢                  ∏                  i          =          1                n            ⁢                          ⁢              N        bi            where Nbi is the multiplicity of the branching element i.
Tomalia in Aldrichimica Acta, Volume 37 Number 2, pages 39-57, 2004, illustrates the mathematical relationship for calculating the number of terminal groups on the surface of the last generation, the number of covalent bonds formed up to the G generation, called “number of branching cells”, BC, and molecular weight PM, for a dendrimer with a multiplicity nucleus Nc, and a branching unit Nb.
      BC    =                  N        c            ⁡              [                                            N              b              G                        -            1                                              N              b                        -            1                          ]                  PM    =                  M        c            +                        N          c                ⁡                  [                                                    M                RU                            ⁡                              (                                                                            N                      b                      G                                        -                    1                                                                              N                      b                                        -                    1                                                  )                                      +                          MN              b              G                                ]                    Mc Represents the molecular weight of the nucleusMRU Represents the Branching UnitM Represents the Surface Terminal GroupAdditionally, the amount of covalent bonds in generation G, indicated by CG, is given in the equation:CG=NcNbG-1;G≧1
In Table 1, there are examples of the numbers of the terminal groups of generation 0 to 10, for two dendrimers with different nucleus multiplicity.
TABLE 1Comparison of the number of functional groups for a nucleus withmultiplicities of 3 and 4.TrifunctionalTetrafunctionalnucleusnucleusNc = 3Nc = 4Number ofNumber ofterminal groupsterminal groupsGenerationfor Nb = 2for Nb = 203416821216324324486459612861922567384512876810249153620481030724096
The plurality of functional groups on the periphery of a dendrimer, is one of the most important characteristics, and the reason for which they are employed in various fields of application, such as medical diagnosis, reported in Chemical & Engineering News, Jun. 13, 2005, pages 30-36; Wiener et al., Magnetic Resonance. in Medicine I, 1994, 31, pages 1-8; Adam et al., Magnetic Resonance in Medicine, 1994, 32, pages 622-628; vectors for DNA reported by Dufes et al., Advanced Drug Delivery, 2005, 57, pages 2177-2202; Bielinska et al., Bioconjugate Chemistry; 10 (5); pages 843-850, 1999; Kim et al., Biomacromolecules; volume 5 (6); pages 2487-2492, 2004 and controlled release of drugs such as reported by Ihre et al., Bioconjugate Chemistry, volume 13, pages 443-452, 2002; Jevprasesphant et al., Journal of Pharmacy and Pharmacology, 2005, volume 56; Patri et al., Bioconjugate Chemistry; volume 15 (6); pages 1174-1181, 2004; Ambade et al., Mollecular Pharmaceutics, volume 2 (4), pages 264-272, 2005. However, reports on using the great number of branching points and covalent bonds formed by arriving at a certain dendrimer generation have not been found.
Frechet et al., report internal modification of a dendrimer via the use of different structural elements to make fine adjustments to the microenvironment of the dendrimer, Journal of Organic Chemistry; volume 65 (22), pages 7612-7617, 2000.
A recent revision by Duncan et al., Advanced Drug Delivery Reviews, Volume 57 (2005) 2215-2237, on the biocompatibility and toxicity of dendrimers, presents strategies that have been employed to date to utilize the dendritic topology on nanodevices for medical purposes and in which is shown that the strategy presented in this invention has not yet been explored. The great majority of the reports on dendrimer synthesis for various fundamental and application studies, only use chemical modification of the nucleus or of the periphery of the structure and the branching structures as support for the connection between both parts and their use for other purposes has received little attention. When the functional groups on the periphery are used, the introduction of various molecules with specific purposes, yet different from each other, such as those indicated in the references cited in this document, it is in detriment to the quantities that it becomes possible to introduce the others, since all of them utilize the terminal groups of the dendrimer. This commitment requires, in some cases, the use of dendrimers with high generations (having a great number of terminal groups) in order to have sufficient active molecules for the effect and properties desired, that in some cases are key. Therefore, the loading capacity of any given dendrimer will be limited to a maximum corresponding to the number of terminal functional groups. In FIG. 2, a dendrimer of this type is shown in diagrammatic form illustrating examples of its components for a biomedical application. The codes in FIG. 2 are given as stars, representing their load, circles represent solubilizing terminal groups, triangles represent routing terminal groups, points represent branching elements, and lines represent spacers.
It is possible to extend the strategies for building dendrimers and dendrons if one visualizes the branching structure as being made up by two or more parts, a functional structure (for example, a drug), hereinafter called load, and the structures that serve as spacers, and structures that provide the point of branching for forming the following one. This allows the inclusion of bifunctional molecules, at least, on each arm being built before coupling a molecule that serves as a branching element.
The possibility of introducing active molecules from the first generation or directly joined to the nucleus, will avoid the partial annexation that is achieved in high generations caused by being sterically hindered induced by the proximity of the surface functional groups in conventional dendrimers and dendrons, mainly with voluminous molecules; the example of spacer molecules is useful in both situations. Thus, to cite an example of this, Khandare et al, Bioconjugate, Chemistry; 16 (2), pages 330-337; 2005, only achieved 32% yield from the terminal groups in the conjugation of the steroid, methylprednisolone (as the load) with a fourth generation PAMAM dendrimer with 64 terminal hydroxyl groups. This is the equivalent of 20 steroid molecules. This is a situation that would be possible with a second generation dendrimer under the strategy of this invention.
In this way, this invention presents dendrimer structures with the type of characteristics that make use of the internal structure of the dendrimer being built to place additional molecules via covalent bonds between elements characteristic of amplifying a certain dendrimer.